Antibodies against T cells as therapeutics

ABSTRACT

A pharmaceutical kit and process useful for achieving prolonged immunosuppression and tumor cell elimination, wherein the kit comprises a first antibody having binding specificity for T cells and is capable of eliminating T cells in vivo; and a second antibody, having binding specificity for T cells and is capable of eliminating T cells in vivo, capable of modulating the antigen effect of T cells or both, wherein said first antibody differs from said second antibody in the constant region of its heavy chains and thus belongs to a different animal species, wherein said first antibody and said second antibody are maintained separately in said kit, and wherein in said process said first antibody is first applied once or several times and said second antibody is applied at a different time from said first antibody.

This is a National Stage filing of PCT/EP95/01898, filed May 19, 1995.

FIELD OF THE INVENTION

The present invention relates to antibodies against T cells which areuseful as therapeutic agents for prolonging immunosuppression and fortumor cell elimination.

BACKGROUND OF THE INVENTION

Heretofore, transplant rejection has been treated with immunosuppressantagents, e.g., monoclonal, immunosuppressive antibodies against human Tlymphocytes which have been generated from mice, rats or goldenhamsters. However, the effect of these antibodies is limited, since thepatient develops an immunoreaction to antibodies which are derived fromanother animal species. This results in what are called antiantibodies,which inhibit the immunosuppressive effect of the injected monoclonalantibodies. Thus, at present, e.g., patients with kidney transplantsthat suffer from transplant rejection crises, are usually treated withonly a single antibody therapy. If another rejection crisis occurs, thistreatment is usually not repeated because of the possible formation ofantiantibodies.

So far, there is no clinical therapy of choice for prolonging theimmunosuppressive effect of antibodies while avoiding the formation ofantiantibodies. A repeated treatment with another monoclonal antibodycan lead to an accelerated formation of antiantibodies (Chatenoud,Transpl. Proc., 25, 2(Suppl. 1):68 (1993)). In addition, patients havedeveloped antiantibodies even against immunosuppressive antibodies thathad been humanized using genetic engineering methods, i.e., where theantibodies have been substantially adapted to the patient's species,i.e., "primate species" or "species-adapted" antibodies (Isaacs et al,Lancet., 340:748 (1992)).

Experimentally, a clear prolongation of the survival time of skintransplants has been found in a mouse model, which was considered atolerance induction. Such prolongation was observed after the injectionof high doses of a rat antibody directed to mouse T(L3T4+Lyt-2) cells,followed by injection of a second antibody of the same species and thesame cell binding specificity, which, however, differed from the firstantibody by its low elimination of T cells from the blood circulation ofthe mouse (a "non-depleting", i.e., eliminating antibody). Unlike thepresent invention, the described principle of action therein was notbased on a combined therapy of at least two antibodies withspecies-different Fc regions (Cobbold et al, Eur. J. Immunol., 20:2747(1990)).

Prolonged survival time of skin transplants and lack of formation ofantiantibodies, were also found after the injection of a rat anti-mouseT(L3T4=CD4+lymphocyte subpopulation)-cell antibody, followed byinjection of (Fab')₂ fragments and unfragmented monoclonal hamsteranti-mouse T(CD3) antibodies (Hirsch et al, Transplantation, 47:853(1989)). Here, too, the described principle of action is not based on acombined therapy of two antibodies having species-different Fc regionsthat are directed to all T cells, as in the present invention, but,rather, on the suppression of the CD4+T lymphocyte subpopulation (Hirschet al, J. Immunol., 140:3766 (1988)) achieved by means of the firstantibody, which is, however, not sufficient.

Permanent tolerance of skin transplants can be achieved in irradiatedmice after transplantation of bone marrow of the donor of the skintransplant, while protecting anti-T cell antibodies (Thierfelder et al,Blood, 68:818 (1986)). However, this technique involves risks.

So far, there is no therapy of choice for definitely preventing theformation of antiantibodies in a patient in the case of conventionalpoly- or monoclonal immunosuppressive antibodies. The first clinicalexperiences with antibodies that have recently been humanized by meansof genetic engineering show that antiantibodies may be formed (Isaacs etal, supra) similarly to what was seen with murine immunosuppressiveanti-mouse T cell antibodies (Kremmer et al, Eur. J. Immunol., 23:1017(1993)).

Also, a combination of immunosuppressive antibody treatment withchemotherapy, e.g., cyclophosphamide or busulfan, involves the risk ofside-effects, particularly on hematopoiesis, and, also, on thetransplanted tissue, due to lack of cell specificity of thechemotherapeutic agents (Cobbold et al, supra; and Leong et al, Eur. J.Immunol., 22:2825 (1992)).

SUMMARY OF THE INVENTION

An object of the present invention is to provide antibodies for clinicaltherapy for prolonging the immunosuppressive antibody effect, whileavoiding the formation of antiantibodies.

The above-described object of the present invention has been met by theuse of antibodies against T cells as a therapeutic for prolongedimmunosuppression and tumor cell elimination, wherein the antibodiesconsist of at least two different groups A, B, which are administered atdifferent times and in which at least one antibody type of group Bdiffers from at least one antibody type of group A in the constantregions of their heavy chains, and wherein group A, which is firstapplied once or several times, has a T-cell eliminating effect, whereasthe other group B (which is applied at a different time) has a T-celleliminating and/or T-cell antigen modulating effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing prolonged immunosuppression due to sequentialtreatment with two anti-T cell antibodies, the Fc regions of which arespecies-different.

FIG. 2 is a graph showing immunosuppression using anti-T cell antibodieswith mouse or rat Fc regions.

FIG. 3 is a table showing suppression of antiantibodies byFc-region-incompatible monoclonal antibody therapy.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the above-described objects of the present inventionhave been met by the use of antibodies against T cells as a therapeuticfor prolonged immunosuppression and tumor cell elimination, wherein theantibodies consist of at least two different groups A, B, which areadministered at different times and in which at least one antibody typeof group B differs from at least one antibody type of group A in theconstant regions of their heavy chains, and wherein group A, which isfirst applied once or several times, has a T-cell eliminating effect,whereas the other group B (which is applied at a different time) has aT-cell eliminating and/or T-cell antigen modulating effect.

Preferably, antibody B is a bispecific antibody.

Also, preferably, antibody B has a different constant region in itsheavy chains than antibody A, and antibody A or antibody B is obtainedby genetic engineering of the region encoding the constant region of itsheavy chain.

Also, preferably, antibody A or antibody B is a humanized antibody.

Also, preferably, antibody A or antibody B has a hapten covalentlylinked to the constant region of its heavy chain. The hapten ispreferably DNP or TNP.

Each of the two groups of antibodies, i.e., antibodies A and antibodiesB, may also consist of only one antibody type or several kinds ofantibodies.

In addition, the two groups of antibodies, i.e., antibodies A andantibodies B, may also be monoclonal or polyclonal.

The sequential treatment with anti-T cell antibodies that are partiallyor fully humanized using molecular biological means, and non-humanizedantibodies, or with at least two anti-T cell antibodies generated fromdifferent species, as described herein, leads to prolongedimmunosuppression and tumor cell elimination. This treatment principlehas been experimentally tested on animals, as shown below, and has notheretofore been described in the art.

The novel therapy principle of the present invention, which has notheretofore been described in the art, is not obvious at all in terms ofimmunology. Commonly, immunobiologists and experts in the field ofmedicine, search for a reduction of the immunogenicity of anti-T cellantibodies (which causes the formation of antiantibodies) by adaptationthereof, as much as possible, to the patient's antibody immunoglobulinstructures so that the patient is more likely to tolerate theantibodies, e.g., by humanization by means of genetic engineering ofmonoclonal immunosuppressive antibodies derived from mice. However,principally this adaptation cannot possibly be complete, and is thecause for the formation of antiantibodies, because the T cell binding(V) region of the immunosuppressive antibody is so variable that thepatient's immunoapparatus can still form antiantibodies thereto.

The present invention is based on a contrasting experience, i.e., on thesuppression of antiantibodies by creating a high species differencebetween the anti-T cell antibodies, wherein one or both, applied alone,may be potentially immunogenic in the recipient of the antibodies.

It was found in the present invention that:

(a) the survival time of skin transplants was basically prolonged, notby applying two different mouse anti-mouse T cell antibodies (or twodifferent rat anti-mouse T cell antibodies) one after the other, but byusing two antibodies which are species-different to one another, but notnecessarily to the recipient of the antibodies, and a clear prolongedimmunosuppression was achieved; and

(b) two antibodies are effective even when they are as different fromone another as human and mouse.

These thoughts, results and antibody combinations define a therapy modeloffering, inter alia, the advantage that it can immediately be testedclinically, and does not expose the patients to any additional treatmentrisks. A prolongation of immunosuppression should not only be a moresuccessful therapy for rejection crises of organ transplants and immunecomplications with bone marrow transplantations, but should help preventthem altogether by prophylactic treatment. In addition, autoimmunediseases, chronic diseases of all kinds of rheumatism, and alsoindividual tumor conditions might face new therapeutic perspectives. Forinstance, in the mouse model studies carried out by the inventors on thesuppression of murine or human T cell leukemias transplanted into mice,a prolonged survival time due to antibody injection was observed. Upon Tcell depletion, foreign immunocompetent cells can be introduced intochimeric mice, i.e., mice transplanted with bone marrow and sufferingfrom leukemia, which foreign immunocompetent cells attack the neoplasticcells in the recipient. Furthermore, the tolerance induction, vis-a-visheterologous serum proteins makes possible passive vaccination withantibodies of a different species that is free of hypersensitivereactions, e.g., for tetanus.

In the murine skin transplant model it could be shown that a monoclonal,immunosuppressive antibody, that was humanized by genetic engineeringmethods achieves a survival time of transplants against murine Tlymphocytes, i.e., prolonged by a multitude, when its application waspreceded by one or more injections of a monoclonal immunosuppressivemouse antibody. The preceding antibody injections as such did not haveto be immunosuppressive in the sense of transplant prolongation. Itturned out that this antibody therapy induced a complete tolerancetowards heterologous, human serum protein, which still remained fivemonths after the end of the immunosuppressive therapy. The unexpectedprolongation of the immunosuppressive effect was thus, accompanied by alack of a formation of antiantibodies in the treated mice due to theirtolerance of the heterologous antibody immunoglobulin. The principle ofaction underlying this phenomenon is analyzed particularly with regardto species-related differences in the Fc region of the combinedantibodies. It also proved effective when using anti-T cell antibodiesthat had not been modified by molecular biology, if they werespecies-different from one another.

Antibodies have what is called a variable region that includes theantibody binding site and what is called a constant Fc region thatmediates antibody effector functions (e.g., elimination from the systemof body cells occupied by antibodies), which is located on what arecalled the constant regions of the heavy chains of the antibody. In thisway, two antibodies can be similar with regard to their specificity tobind, e.g., human T lymphocytes. Such antibodies with the same cellbinding specificity, however, may differ in their Fc region due to thefact that they are derived from different normal or molecularbiology-manipulated animal species. They can also be modified in vitroin the Fc region using methods of molecular biology on generatingantibody-secretory cells (e.g., hybridomas or hybrid hybridomas) so thatthere is the degree of difference obtained as found between humans androdents, and as described in the present invention.

In the following examples, the present invention is described in moredetail.

EXAMPLE 1

Combination antibody treatment was carried out by first injecting amouse IgG_(2a) anti-mouse-Thy-1.2 antibody (MmT1 antibody; (Kremmer etal, supra)) on day 3, followed by injecting a chimeric antibody having aMmT1 idiotype (V region) and human Fc IgG₁ region (T23 antibody) on day0 and twice a week. The T23 antibody differs from the MmT1 antibody byan exchange of the murine IgG, Fc region for a human IgG₁ Fc region,which was achieved by means of genetic engineering. The results areshown in FIG. 1.

As shown in FIG. 1, a single dose of MmT1 did not prolong the (average)skin survival time. T23 alone, applied twice a week, did prolong it bynine days from 16 to 24. MmT1 (first dose) followed by T23 (appliedtwice a week) prolonged it to more than 90 days. Thus, FIG. 1 showsimmunosuppression that was prolonged approximately ten-fold, as measuredin a rodent skin transplantation model of maximum histoincompatibilty,by the combined Fc-region incompatible antibody treatment of the presentinvention.

Also, as shown in FIG. 1, a similarly increased immunosuppression wasachieved after replacement of antibody MmT1 with antibody MmT5 (Kremmeret al, supra), which does not differ from antibody MmT1 in its T-cellspecificity, but, rather, differs in the microstructure of the antibodybinding site (idiotype).

The results demonstrate that in the combined Fc-region incompatibleantibody therapy of the present invention, the likeness or difference ofthe antibody binding site is not a prerequisite for the principle ofaction, but, rather, the species-dependent difference of its heavychains incorporating the Fc regions is a prerequisite for the principleof action.

EXAMPLE 2

MmT1/RmCD4+CD8 combination therapy was carried out by injecting MmT1 onday 3, followed by injecting RmCD4+RmCD8 antibody (a rat anti-mouseCD4+CD8 lymphocyte antibody) on day 0 and twice a week, and vice versa.The results are shown in FIG. 2.

FIG. 2 shows that rat anti-mouse T cell antibodies having Thy-1specificity (Kummer et al, J. Immunol., 138:4069 (1987)), and alsoparticularly clinically-relevant antibody specificities, such asanti-CD4 and anti-CD8 (two T cell subpopulations, which together bindall T cells) also prolong the average survival time of skin transplants.Furthermore, as shown therein, the reversal of the combined antibodytreatment in the RmCD4+CD8/MmT1 combination also leads to a prolongedimmunosuppression since here, too, the prerequisite of thespecies-dependent difference of the Fc region is fulfilled. Sinceanti-CD4+CD8 antibodies are active as first antibodies, this excludes aneffect of the preinjected first antibody restricted to MmT1. On thecontrary, the prerequisite for the synergistic antibody action of theirspecies-dependent Fc region differences (apart from the application ofat least two antibodies at different times) applies again and again.Survival of skin transplants using a combination antibody therapy waspermanent if further T cell depleting and/or T cell receptor modulating(anti-CD3) antibodies were added to the second antibody.

EXAMPLE 3

Groups of 4 to 6 C57BL/6 mice were injected with 400 μg of the firstantibody (shown in FIG. 3) and 500 μg of the second antibody (shown inFIG. 3). Tail blood was drawn 6 to 10 days after the last injection inorder to determine the antiantibody level. The results are shown in FIG.3.

As shown in FIG. 3, the combined rat/mouse or mouse/rat Fc-regionincompatible antibody treatment leads to a high suppression or completelack of the formation of antiantibodies, i.e., antiantibody levels wereextremely low or zero where there was a species difference (rat/mouse ormouse/rat) between the first and the second antibody. The same appliesto treatment when carried out as per Example 1.

Antiantibodies also occur when treating with polyclonal antibodies thatarise after immunization of, e.g., rabbit, rat or horse lymphocytes.Here, too, it can be seen that a species difference (e.g., rabbit/rat)of polyclonal antibodies leads to a prolonged immunosuppression in mice,as well as with what are called bispecific antibodies, i.e., antibodieshaving two different binding sites, or with anti-T cell antibodies thatwere chemically modified by introduction of a low-molecular compound(e.g., DNP, TNP haptenes) or by genetic engineering, e.g., antibodiesand antibody fragments prepared in bacteria. Here, too, sequentiallyinjected anti-T cell antibodies may neutralize the formation ofantiantibodies to species-different polyclonal or bispecific orchemically or molecular biology-modified antibodies. A prerequisite isalways a strong difference in the sequentially applied antibodies orantibody groups, which either results from species difference or fromthe introduction (conjugation) of chemical compounds.

Finally, undesired immunoreactions may also occur in the case of passiveimmunization with antibodies in protein-oversensitive or presensitizedpatients. A treatment using combined Fc-region incompatible antibodytherapy would prevent the formation of antiantibodies.

What is claimed is:
 1. A combination of pharmaceutical compositionsuseful for achieving prolonged immunosuppression and tumor cellelimination, comprising:(A) a first pharmaceutical compositioncomprising a first antibody having binding specificity for T cells andwhich is capable of eliminating T cells in vivo; and (B) a secondpharmaceutical composition comprising a second antibody having bindingspecificity for T cells and which is capable of eliminating T cells invivo, or is capable of modulating the antigen effect of T cells or isboth capable of eliminating T cells in vivo and capable of modulatingthe antigen effect of T cells,wherein said first antibody has adifferent constant region in its heavy chains than said second antibody,and belongs to a different animal species than said second antibody,wherein said first pharmaceutical composition and said secondpharmaceutical composition are maintained separately in said kit.
 2. Thecombination of pharmaceutical compositions as claimed in claim 1,wherein said second antibody is a bispecific antibody capable of bindingto two different antigenic determinants.
 3. The combination ofpharmaceutical compositions as claimed in claim 1, wherein one of saidanimal species is human.
 4. The combination of pharmaceuticalcompositions as claimed in claim 1, wherein said first antibody or saidsecond antibody is obtained by genetic engineering of the regionencoding the constant region of its heavy chain.
 5. The combination ofpharmaceutical compositions as claimed in claim 4, wherein said firstantibody or said second antibody is a humanized antibody.
 6. Thecombination of pharmaceutical compositions as claimed in claim 1,wherein first antibody or said second antibody has a hapten covalentlylinked to said constant region of its heavy chain.
 7. The combination ofpharmaceutical compositions as claimed in claim 6, wherein said haptenis selected from the group consisting of DNP and TNP.
 8. The combinationof pharmaceutical compositions as claimed in claim 1, wherein at leastone of said first antibody or said second antibody is a polyclonalantibody.
 9. The combination of pharmaceutical compositions as claimedin claim 1, wherein at least one of said first antibody or said secondantibody is a monoclonal antibody.